The goal of Project 1 is to study the roles and relationships between the homeobox-containing genes Msx2 and D1x-5 in regulation of pattern formation and cartilage differentiation in the developing chick limb bud. msx2 and D1x-5, members of the Msh and Distal-less families, are co-expressed in the apical ectodermal ridge (AER) of the limb bud, which plays a crucial role in directing outgrowth and patterning of the lib, and are also co-expressed in the anterior mesoderm of the limb bud, which during normal development undergoes little outgrowth and fails to give rise to skeletal structures, suggesting that these genes may be intimately involved in regulatory networks controlling AER activity and specifying the anterior non-skeletal forming region of lib bud mesoderm. To study these possibilities, Msx2 and D1x-5 retroviral expression vectors will be used to examine the effect of localized ectopic expression of these genes, alone and in combination, in the distal anterior ectoderm of the chick wing bud on one another's expression; on their own expression; on the expression of genes for signalling molecules, such as FGF-4, FGF-2, or Wnt-5a that have been implicated in mediating AER activity; and, on morphogenesis and gene expression in the underlying anterior mesoderm. Effects of localized ectopic expression of Msx2 and D1x-5, alone and in combination, in the posterior mesoderm of the limb bud on morphogenesis and gene expression will also be studied. In addition to being expressed in the AER and anterior mesoderm of the limb bud, D1x-5 is expressed at high amounts in the proximal central core of the limb bud at the onset of the chondrogenic differentiation of the skeletal elements of the limb and the initiation of the expression of genes for several cartilage-specific matrix proteins. There is also a strong correlation between D1x-5 expression and the onset of cartilage-specific gene expression in vitro. Dix-5 expression vectors will be used to determine if D1x-5 can promote the expression of genes for cartilage-specific matrix proteins and can regulate expression of reporter constructs containing promoter and enhancer elements of the type II collagen gene. If so, the interaction of D1x-5 protein with putative homeodomain binding sequences in the first intron enhancer element in the type II collagen gene in vitro ad in vivo will be studied. Depending on the outcome of the preceding experiments, studies will be initiated to identify other potential target genes of D1x-5 in differentiating limb cartilage and proteins with which D1x-5 might interact to regulate gene expression in these cells.